This invention relates to a method for the rapid determination of glycosylated proteins in blood and a means for carrying out this method. The accurate assay of glycosylated protein levels in human blood and in particular, the quantity of glycoproteins to be found in the plasma and the fraction of glycosylated hemoglobin found in erythrocytes is important, particularly to those suffering from diabetes, because together they can yield a more accurate medium term and long term picture of an individual's glycemic state compared to blood glucose level which is subject to immediate large fluctuations due to factors such as food intake and physical activity among others. For example, such information is significant in determining the effects of therapy, changes in therapy and lifestyle changes on the extent to which the disease is under control in an individual. In addition, the initial diagnosis of diabetes can be made with more certainty when longer term indicia of an individual's glycemic state are taken into account.
According to information compiled by the National Diabetes Information Clearinghouse (NDIC), (NIH publication No. 96-3926, October, 1995), there were an estimated total of 16 million cases of diabetes in the United States in 1995 of which only about 8 million were diagnosed. The number of people suffering from insulin-dependent diabetes (IDDM) was estimated as high as 800,000, with noninsulin-dependent diabetes (NIDDM) estimated from about 7 to 7.5 million diagnosed cases. The estimated total medical costs directly attributable to diabetes were $45 billion and the estimated indirect costs due to disability, work loss and premature mortality were $47 billion in 1992. Diabetes was the seventh leading cause of death listed on United States death certificates in 1993, according to the National Center for Health Statistics. Based on the period 1990-1992, about 595,000 new cases of NIDDM and about 30,000 cases new cases of IDDM are diagnosed each year. Based on 1993 estimates about 100,000 children aged 19 or younger and 3.2 million adults aged 65 years or older were afflicted with this disease. People afflicted with diabetes also suffer from long term complications that strike them to a much greater degree than the general population. These include: heart disease, stroke, high blood pressure, blindness, kidney disease, nerve disease, amputations, dental disease.
From the above statistics it can be seen that the detection of diabetes and its control, is a health issue of major significance.
According to the NDIC treatment for diabetes emphasizes control of blood glucose through blood glucose monitoring, regular physical activity, meal planning, and attention to relevant medical and psychosocial factors. Oral medications and/or insulin injections are also required by many patients, for glucose control. The NDIC states (NIH publication No. 96-3626, October, 1995): "Treatment of diabetes is an ongoing process that is planned and regularly reassessed by the health care team, the person with diabetes, and his/her family."
A key element in the treatment of diabetes must therefore be the monitoring of the extent to which physiological indicia of diabetes have been brought within normal ranges or ranges considered appropriate to the individual by the health care team.
One such indication of the effectiveness of treatment is blood sugar level. Although commonly used, blood sugar level may not always give an accurate picture of the glycemic state of an individual as it is subject to variations produced by diet and physical activity.
Another approach to assessing the glycemic state of an individual is to determine the level of glycosylated proteins in blood serum and the level of glycosylated hemoglobin. Prolonged elevation of blood glucose in diabetes causes an increase in level of nonenzymatically glycosylated hemoglobin and glycosylated proteins. Glycosylated serum protein levels have been correlated with both fasting serum glucose and glycosylated hemoglobin levels. The degree of glycosylation of hemoglobin and serum proteins has been correlated with indices of glycemia. See for example: Bunn H P, Gabbay K H, Gallop P M, The glycosylation of hemoglobin: relevance to diabetes mellitus. Science 1978; 200:21-27; Dun P J, Cole R A, Soeldner J S et al. Temporal relationship of glycosylated hemoglobin concentration to glucose control in diabetics. Diabetologia 1979; 17:213-220; Paisey, R B, Macfarlane, D G Sherriff R J, Hartog M, Slade R R, White, D A, The relationship between blood glycosylated hemoglobin and home capillary blood glucose levels in diabetics. Diabetologia 1980; 19:31-34; McFarland K F, Catalano E W, Day J F, Thorpe S R, Baynes J W, Nonenzymatic glycosylation of serum proteins in diabetes mellitus. Diabetes 1979; 28: 1011-1014. Yue D K, Moris K S, Turtle J R, Glycosylation of plasma protein and its relation to glycosylated hemoglobin in diabetes, Diabetes 1980; 29:296-300, Dolhofer R, Wieland O H, Glycosylation of serum albumin in diabetes mellitus, Diabetes 1980; 29:417-422.
According to the literature in the field (see the above references) the following indicia can be used for the detection of diabetes in the first place and as measures of the degree to which it has been brought under control in an individual:
Blood serum glucose level: Under properly controlled circumstances this may give a good indication of the presence of the disease and the success of measures to control it in many individuals. The result of such assays in effect is a snap-shot of the glucose level at the time the test was taken. Generally the patient has fasted prior to the test in order to obtain a baseline glucose level. However, in some insulin-dependent diabetics large day-to-day variations in plasma glucose concentrations make this method ineffective.
Degree of non-enzymatic glycosylation of proteins found in blood plasma and serum. This is based on the non-enzymatic reaction of glucose with the available amino groups on proteins, such as albumin found in blood plasma. The chemistry of this process has been well worked out (see the above references): the glucose forms a Schiff base (or aldimine) with the amino group of the protein. The glucose moiety of the Schiff base then rearranges in a relatively slow step to form a fructosamine derivative of the protein (a glycoprotein). Thus, glycoprotein level does not reflect moment to moment changes in blood plasma (or serum) glucose levels, but is a longer term averaged reflection of blood glucose level. It has been established that plasma or serum fructosamine levels are a time averaged reflection of fasting blood glucose levels over the two to three week period prior to administration of the test (see L. Kennedy, et al, Diabetologia(1981) 21: 94-98). It is unaffected by time of day or previous food intake. An overnight fast is not required to yield valid results. Assays of plasma or serum for the fructosamine moiety are generally useful, for example, in assessing the glycemic state of diabetes patients where there has been a change in therapy.
Degree of glycosylation of hemoglobin: According to investigators in the field (e.g. McFarland, et al (Diabetes 1979; 28: 1011-1014)) levels of glycohemoglobins are indicative of the time-averaged blood glucose concentration over a period of several weeks due to the slow rate of glucosylation of hemoglobin and the 120 day average life span of erythrocytes. As with serum proteins, fructosamine derivatives are formed by reaction of available amino groups on hemoglobin with glucose by a non-enzymatic route (see for example Bunn, et al). This test is a measure of glycemic state for the period two to three months prior to the administration of the test. It is known in this field that poorly controlled diabetics may have two to four times the normal glycosylated hemoglobin level. Glycohemoglobin levels are also useful in assessing the level of control in insulin-dependent diabetics that have large day-to-day variations in plasma glucose concentrations.
Current procedures for the determination of glucosylated serum proteins include: a wet chemical method using thiobarbituric acid in which nonenzymatically bound glucose is released as 5-hydroxymethylfurfural and is assayed colorimetrically (see for example, McFarland, et al, Diabetes, vol 28, pp1012-1014, 1979); a wet chemical method utilizing the oxidation of fructosamine under alkaline conditions by nitroblue tetrazolium (NBT) to form a derivative that is assayed detected colorimetrically relative to a standard based on 1-deoxy-1-morpholinofructose (DMFr) (see Johnson, et al, Clinica Chemica Acta, vol. 27, pp 87-95, 1982); Burd et al, in U.S. Pat. No. 5,470,752 disclose a colorimetric method, based on a multilayered device for analyzing the concentration of fructosamine in a liquid sample that utilizes the ability of fructosamines to reduce an indicator such as NBT; Burd et al, in U.S. Pat. No. 5,639,672 disclose electrochemical methods for analysis of fructosamine in biological fluids, including the amperometric detection of the reduction of NBT by fructosamines.
Determination of glycosylated hemoglobins in whole blood, in practice often rely on fractionation procedures such as column chromatography, ion exchange resins and electrophoretic separation. The separation procedure may provide quantitative data on the level of glycosylated hemoglobin or colorimetric procedures may be used to obtain such data (see Isolab, Inc. publication on glycohemoglobin determination, revised Sep. 1, 1986, McFarland, et al, Diabetes, vol. 28, 1011-1014, 1979.). Work on separation of glycosylated hemoglobins from human blood led to the term Hb A.sub.1 (A.sub.1) to characterize all of the glycosylated hemoglobin fractions.
Currently, in the clinical laboratory setting, chromatographic and spectrophotometric instrumentation using either absorption or reflectance of light is generally used to assay blood glycosylated hemoglobin levels. Such instrumentation is expensive and relatively complex to use. The usual turnaround time in the clinical lab is at least 24 hours per analysis and the cost is relatively high. There is a need for a simple, rapid, highly sensitive, accurate and reproducible method to determine glycosylated hemoglobin levels and a means to carryout this method which can be easily miniaturized, inexpensively produced, and which is inexpensive to use. Such a method and means would be especially useful, convenient, and less painful to the patient when screening for and monitoring diabetes in the human if only a drop of blood was required for a reliable rest.
Both glycosylated hemoglobin levels which reflect the relatively long term glycemic state of a diabetic patient, and levels of glycosylated serum or plasma proteins, which reflect more recent, time averaged changes in the diabetic patient's glycemic state, resulting from changes in therapy or other factors, are important for the effective management of the diabetic patient. From the perspective of both the medical practitioner and the diabetic patient, it would be advantageous to have a simple, inexpensive method for determining glycosylated hemoglobin and levels of glycosylated serum or plasma proteins, that is convenient to use and that produces little or no pain and/or inconvenience for the patient. There is a need for a rapid, easy to use, inexpensive method to immediately obtain both the level of glycoproteins in plasma and the level of glycosylated hemoglobins in the red blood cells on a small, undiluted sample of whole blood.
In addition, for the foregoing reasons, there is a need for a device for the quantitative assay of glycoprotein, including glycosylated hemoglobin in blood and other fluids which is simple to use, is highly sensitive, accurate and reproducible and which can be easily miniaturized, inexpensively produced and inexpensively used to carry out such a method.